Modified Bacillus thuringiensis insecticidal-crystal protein genes and their expression in plant cells

ABSTRACT

A DNA fragment, encoding all or an insecticidally-effective part of a Bt crystal protein, is modified by changing A and T sequences to corresponding G and C sequences encoding the same amino acids, in a region having transcriptional activity less than 25% of the transcriptional activity of a region at the translation initiation site.

This application is a divisional of application Ser. No. 08/453,104,filed May 30, 1995 issued as U.S. Pat. No. 5,663,446; which is acontinuation of application Ser. No. 07/937,869, filed as PCT/EP91/00733Apr. 17, 1991, now abandoned.

This invention provides a modified Bacillus thuringiensis ("Bt") gene(the "modified BtICP gene") encoding all or an insecticidally-effectiveportion of a Bt insecticidal crystal protein ("ICP"). A plant,transformed with the modified Bt ICP gene can show higher expressionlevels of the encoded ICP and improved insect-resistance.

BACKGROUND OF THE INVENTION

Plant genetic engineering technology has made significant progressduring the last 10 years. It has become possible to introduce stablyforeign genes into plants. This has provided exciting opportunities formodern agriculture. Derivatives of the Ti-plasmid of the plant pathogen,Agrobacterium tumefaciens, have proven to be efficient and highlyversatile vehicles for the introduction of foreign genes into plants andplant cells. In addition, a variety of free DNA delivery methods, suchas electroporation, microinjection, pollen-mediated gene transfer andparticle gun technology, have been developed for the same purpose.

The major aim of plant transformations by genetic engineering has beencrop improvement. In an initial phase, research has been focused on theengineering into plants of useful traits such as insect-resistance. Inthis respect, progress in engineering insect resistance in transgenicplants has been obtained through the use of genes, encoding ICPs, fromBt strains (Vaeck et al., 1987). A Bt strain is a spore forminggram-positive bacterium that produces a parasporal crystal which iscomposed of crystal proteins which are specifically toxic against insectlarvae. Bt ICPs possess a specific insecticidal spectrum and display notoxicity towards other animals and humans (Gasser and Fraley, 1989).Therefore, the Bt ICP genes are highly suited for plant engineeringpurposes.

For more than 20 years, Bt crystal spore preparations have been used asbiological insecticides. The commercial use of Bt sprays has howeverbeen limited by high production costs and the instability of crystalproteins when exposed in the field (Vaeck et al., 1987). Theheterogeneity of Bt strains has been well documented. Strains activeagainst Lepidoptera (Dulmage et al., 1981), Diptera (Goldberg andMargalit, 1977) and Coleoptera (Krieg et al., 1983) have been described.

Bt strains produce endogenous crystals upon sporulation. Upon ingestionby insect larvae, the crystals are solubilized in the alkalineenvironment of the insect midgut giving rise to a protoxin which issubsequently proteolytically converted into a toxic core fragment ortoxin of 60-70 kDa. The toxin causes cytolysis of the epithelial midgutcells. The specificity of Bt ICPs can be determined by their interactionwith high-affinity binding sites present on insects' midgut epithelia.

The identification of Bt ICPs and the cloning and sequencing of Bt ICPgenes has been reviewed by Hofte and Whiteley (1989). The Bt ICP genesshare a number of common properties. They generally encode insecticidalproteins of 130 kDa to 140 kDa or of about 70 kDa, which contain toxicfragments of 60±10 kDa (Hofte and Whiteley, 1989). The Bt ICP genes havebeen classified into four major groups according to both theirstructural similarities and insecticidal spectra (Hofte and Whiteley,1989): Lepidoptera-specific (CryI), Lepidoptera- and Diptera-specific(CryII), Coleoptera-specific (CryIII) and Diptera-specific (Cry IV)genes. The Lepidoptera-specific genes (CryI) all encode 130-140 kDaproteins. These proteins are generally synthesized as protoxins. Thetoxic domain is localized in the N-terminal half of the protoxin.Deletion analysis of several CryI genes confirm that 3' portions of theprotoxins are not absolutely required for toxic activity (Schnepf etal., 1989). Cry II genes encode 65 kDa proteins (Widner and Whiteley,1985). The Cry II A proteins are toxic against both Lepidoptera andDiptera while the Cry II B proteins are toxic only to Lepidopteraninsects. The Coleoptera-specific genes (Cry III) generally encodeproteins vith a molecular weight of about 70 kDa. (Whiteley and Hofte,1989). The corresponding gene (cry III A) expressed in E. coli directsthe synthesis of a 72 kDa protein which is toxic for the Colorado potatobeetle. This 72 kDa protein is processed to a 66 kDa protein byspore-associated bacterial proteases which remove the first 57N-terminal amino acids (Mc Pherson et al., 1988). Deletion analysisdemonstrated that this type of gene cannot be truncated at its 3'-endwithout the loss of toxic activity (Hofte and Whiteley, 1989). Recently,an anti-coleopteran strain, which produces a 130 kDa, protein has alsobeen described (European patent application ("EPA") 89400428.2). The cryIV class of crystal protein genes is composed of a heterogenous group ofDiptera-specific crystal protein genes (Hofte and Whiteley, 1989).

The feasibility of generating insect-resistant transgenic crops by usingBt ICPs has been demonstrated. (Vaeck et al., 1987; Fischoff et al.,1987 and Barton et al., 1987). Transgenic plants offer an attractivealternative and provide an entirely new approach to insect control inagriculture which is at the same time safe, environmentally attractiveand cost-effective. (Meeusen and Warren, 1989). Successful insectcontrol has been observed under field conditions (Delannay et al., 1989;Meeusen and Warren, 1989).

In all cases, Agrobacterium-mediated gene transfer has been used toexpress chimaeric Bt ICP genes in plants (Vaeck et al., 1987; Barton etal., 1987; Fischoff et al., 1987). Bt ICP genes were placed under thecontrol of a strong promoter capable of directing gene expression inplant cells. It is however remarkable that expression levels in plantcells were high enough only to obtain insect-killing levels of Bt ICPgenes when truncated genes were used (Vaeck et al., 1987; Barton et al.,1987). None of the transgenic plants containing a full-length Bt ICPgene produced insect-killing activity. Moreover, Barton et al. (1987)showed that tobacco calli transformed with the entire Bt ICP codingsequence became necrotic and died. These results indicate that the BtICP gene presents unusual problems that must be overcome to obtainsignificant levels of expression in plants. Even, when using a truncatedBt ICP gene for plant transformation, the steady state levels of Bt ICPmRNA obtained in transgenic plants are very low relative to levelsproduced by both an adjacent NPT II-gene, used as a marker, and by otherchimeric genes (Barton et al., 1987; Vaeck et al., 1987). Moreover, theBt ICP mRNA cannot be detected by northern blot analysis. Similarobservations were made by Fischoff et al. (1987); they reported that thelevel of Bt ICP mRNA was much lower than expected for a chimeric geneexpressed from the CaMV35S promoter. In other words, the cytoplasmicaccumulation of the bt mRNA, and consequently the synthesis, theaccumulation and thereby the expression of the Bt ICP protein in plantcells, are extremely inefficient. By contrast, in microorganisms, it hasbeen shown that truncated Bt ICP genes are less favorable thanfull-length genes (Adang et al., 1985), indicating that the inefficientexpression is solely related to the heterologous expression of Bt ICPgenes in plants.

The problem of obtaining significant Bt ICP expression levels in plantcells seems to be inherent and intrinsic to the Bt ICP genes.Furthermore, the relatively low and poor expression levels obtained inplants appears to be a common phenomenon for all Bt ICP genes.

It is known that there are six steps at which gene expression can becontrolled in eucaryotes (Darnell, 1982):

1) Transcriptional control

2) RNA processing control

3) RNA transport control

4) mRNA degradation control

5) translational control

6) protein activity control

For all genes, transcriptional control is considered to be of paramountimportance (The Molecular Biology of the Cell, 1989).

In European patent publications ("EP") 385,962 and 359,472, efforts tomodify the codon usage of Bt ICP genes to improve their expression inplant cells have been reported. However, wholesale (i.e., non-selective)changes in codon usage can introduce cryptic regulatory signals in agene, thereby causing problems in one or more of the six steps mentionedabove for gene expression, and thus inhibiting or interfering withtranscription and/or translation of the modified foreign gene in plantcells. For example, changes in codon usage can cause differential ratesof mRNA production, producing instability in the mRNA, so produced(e.g., by exposure of regions of the mRNA, unprotected by ribosomes, toattack and degradation by cytoplasmic enzymes). Changes in codon usagealso can inadvertently cause inhibition or termination of RNA polymeraseII elongation on the so-modified gene.

SUMMARY OF THE INVENTION

In accordance with this invention is provided a process for modifying aforeign gene, particularly a Bt ICP gene, whose level and/or rate ofexpression in plant cells, transformed with the gene, is limited by therate and/or level of nuclear production of an mRNA encoded by the gene;the process comprises the step of changing adenine and thymine sequencesto corresponding guanine and cytosine sequences encoding the same aminoacids in a plurality of translational codons of the gene that wouldotherwise directly or indirectly cause a nuclear event which wouldnegatively control (i.e., inhibit or interfere with) transcription,nuclear accumulation and/or nuclear export of the mRNA, particularlytranscription, quite particularly elongation of transcription by RNApolymerase II of the plant cells. Preferably, the adenine and thyminesequences are changed to cytosine and guanine sequences in translationalcodons of at least one region of the gene which, during transcription,would otherwise have thereon a relatively low percentage of RNApolymerase II as compared to another adjacent upstream (i.e., 5') regionof the gene.

Also in accordance with this invention is provided the modified Bt ICPgene resulting from the process.

Further in accordance with this invention, a process is provided forimproving the resistance of a plant against insect pests by transformingthe plant cell genome with at least one modified Bt ICP gene.

This invention also relates to a chimaeric gene that can be used totransform plant cells and that contains the following operably-linkedDNA fragments in the same transcriptional unit:

1) the modified Bt ICP gene;

2) a promoter suitable for directing transcription of the modified BtICP gene in the plant cells; and

3) suitable transcript 3' end formation and polyadenylation signals forexpressing the modified Bt ICP gene in the plant cells.

This invention further relates to:

a cell of a plant, the nuclear genome of which has been transformed tocontain, preferably stably integrated therein, the modified St ICP gene,particularly the chimaeric gene;

cell cultures consisting of the plant cell;

a plant which is regenerated from the transformed plant cell or isproduced from the so-regenerated plant, the genome of which contains themodified Bt ICP gene, particularly the chimaeric gene, and which showsimproved resistance to insect pests;

seeds of the plant; and

a vector for stably transforming the nuclear genome of plant cells withthe modified Bt ICP gene, particularly the chimaeric gene.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "Bt ICP" should be understood as an intact protein or apart thereof which has insecticidal activity and which can be producedin nature by B. thuringiensis. A Bt ICP can be a protoxin, as well as anactive toxin or other insecticidal truncated part of a protoxin whichneed not be crystalline and which need not be a naturally occurringprotein. An example of a Bt ICP is a Bt2 insecticidal crystal protein(Hofte et al., 1986), as well as its insecticidally effective partswhich are truncated at its C- and/or N-terminal ends towards itstryspsin cleavage site(s) and preferably having a molecular weight of60-80 kDa. Other examples of Bt ICPs are: Bt2, Bt3, Bt4, Bt13, Bt14,Bt15, Bt18, Bt21, Bt22, Bt73, Bt208, Bt245, BtI260 and BtI109P asdisclosed in PCT publications WO90/15139 and WO90/09445, in Hofte andWhiteley (1989) and in EPA 90403724.9.

As used herein, "protoxin" should be understood as the primarytranslation product of a full-length gene encoding a Bt ICP.

As used herein, "toxin" or "active toxin" or "toxic core" should all beunderstood as a part of a protoxin which can be obtained by protease(e.g., by trypsin) cleavage and has insecticidal activity.

As used herein, "truncated Bt gene" should be understood as a fragmentof a full-length Bt gene which still encodes at least the toxic part ofthe Bt ICP, preferentially the toxin.

As used herein, "modified Bt ICP gene" should be understood as a DNAsequence which encodes a Bt ICP, and in which the content of adenine("A") and thymine ("T") has been changed to guanine ("G") and cytosine("C") in codons, preferably at least 3, in at least one region of theDNA sequence without affecting the original amino acid sequence of theBt ICP. Preferably in at least two regions, especially in at least threeregions, of the DNA sequence, the A and T content is changed to G and Cin at least 3 codons. For regions downstream of the translationinitiation site of the DNA sequence, it is preferred that the A-Tcontent of at least about 10 codons, particularly at least about 33codons, be changed to G-C.

By "region" of a modified Bt ICP gene is meant any sequence encoding atleast three translational codons which affect expression of the gene inplants.

In accordance with this invention, it has been shown by means of mRNAturn-over studies that the expression pathway of a Bt ICP gene, such asbt2, bt14, bt15 and bt18, is specifically inhibited at the nuclear levelin plant cells. In a further analysis, nuclei of transgenic tobaccoplants, i.e., N28-220 (Vaeck et al., 1987), were used in a nuclearrun-on assay to determine the distribution and the relative efficiencyof RNA polymerase II complexes to initiate transcription of chimaeric BtICP plant genes. In this regard, the run-on assay has been used todetermine initially the relative efficiency of RNA polymerase IIcomplexes to initiate transcription of Bt ICP genes and thereafter todetermine the relative distribution and migration efficiency of the RNApolymerase II complexes on the Bt ICP genes.

N28-220 contains the bt884 fragment under control of the TR 2' promoteras a chimaeric gene. Bt884 is a 5' fragment of the bt2 gene (Hofte etal., 1986) up to codon 610 (Vaeck et al., 1987). Using nuclear run-onanalysis, isolated nuclei of N28-220 were incubated with highly labeledradioactive RNA precursors, so that the RNA transcripts beingsynthesized at the time became radioactively labeled. The RNA polymeraseII molecules caught in the act of transcription in the cell continueelongating the same RNA molecules in vitro.

The nuclear run-on assays of nuclei of N28-220 culture (non-inducedcells and induced cells, TR1'-neo, TR2'-bt884) revealed thattranscription from the TR1' and TR2' promoters is about equallyefficient. This implies that the low Bt ICP (i.e., Bt884) expressionlevels are not due to a specifically reduced transcriptional activity ofthe TR2' promoter. However, nuclear run-on analysis with N28-220 nucleiindicated that transcription elongation of the nascent Bt ICP mRNA isimpaired somewhere between 700 to 1000 nucleotides downstream of thestart of transcription. This means that RNA polymerase II is not able totranscribe the Bt ICP coding sequence with 100% efficiency. Filterbinding assays using labeled Bt DNA fragments spanning this region andprotein extract prepared from tobacco nuclei reveal that this DNA regionundergoes specific interactions with proteins present in nuclei. Theseinteractions are the prime candidates that cause or affect the impairedelongation of transcription by RNA polymerase II through this region. Bymodification of this region to abolish specific protein binding, Bt ICPexpression levels will increase. However, other mechanisms responsiblefor impaired elongation in this region cannot be excluded.

Further in accordance with this invention, sequences within the codingregion involved in negative control of cytoplasmic Bt ICP mRNA levelshave been identified by deletion analysis. To this end, 24 deletionderivatives of pVE36 have been constructed. Three main types of deletionmutants have been constructed (see FIG. 3):

5' end deletions

3' end deletions

internal deletions.

The expression of a mutant hybrid bt2-neo gene (encoding a fusionprotein of Bt2 (Hofte et al., 1986) and NPTII) has been studied by meansof transient expression experiments using the cat gene as a reference.To this end, the neo mRNA levels were measured in relation to cat mRNAlevels in RNA extracts of SR1 protoplasts. The ratio between the neo andcat mRNA level was used to quantify on a relative basis the nptIItranscript (i.e., mRNA) levels produced by the different constructions.These experiments show that progressive deletions of thecarboxy-terminal (i.e., 3') part or the amino-terminal (i.e., 5') partof the Bt ICP coding sequence result in a gradual increase of the nptIItranscript level. Furthermore, since the changes in transcript levelsare not very abrupt, these results suggest that the low transcriptlevels produced by Bt ICP genes are not controlled by a single factor.Nevertheless, individual modifications of bt2 coding sequence cansignificantly reduce the interference and/or inhibition of theexpression of the mRNA encoded by Bt ICP genes in plant cells at thelevel of transcript elongation, nuclear accumulation and nuclear export.The modification(s) may also affect cytoplasmic regulation andmetabolism of such mRNAs and their translation.

Deletion analysis clearly indicates that several internal sequences,located within the Bt ICP coding region, might be involved in thenegative regulation of the Bt ICP expression. By way of example, a 326bp region (FIG. 6b) was identified in the bt2 gene that is involved inthe negative control of BT ICP expression and that is located betweennucleotide position 674 and nucleotide position 1000, particularly a 268bp region between nucleotide positions 733 and 1000, quite particularlya 29 bp region between nucleotide positions 765 and 794 which carriestwo perfect CCAAT boxes which are known to be able to cause a reductionin elongation efficiency and termination of transcription by RNApolymerase II in animal systems (Connelly and Manley, 1989). Thisinternal gene fragment or inhibitory zone may itself comprise aplurality of inhibitory zones which reduce Bt ICP expression levels orwhich interact directly or indirectly with other zones to inhibit orinterfere with expression. Codon usage of this inhibitory zone has beenmodified in a second step by substituting A-T with G-C without affectingthe amino acid sequence. In this regard, this internal 326 bp fragment(FIG. 6b) has been replaced with a modified Bt ICP fragment of thisinvention containing 63 modified codons. The effect of such modificationof this inhibitory zone on Bt ICP expression has been analyzed both intransient and stable plant transformants. The results show that suchmodification of codon usage causes a significant increase of Bt ICPexpression levels and hence improved insect-resistance.

In addition, N-terminal deletion mutants of the bt2 gene have been madeby deleting the first N-terminal 28 amino acids (Hofte et al., 1986). Itis known for the bt2 gene that the first 28 codons can be deletedwithout loss of toxicity (Hofte et al., 1986; Vaeck et al., 1987). Also,codon usage for three codons, 29 to 31, has been changed in accordancewith this invention by replacing A-T with G-C without affecting theamino acid sequence. Furthermore, an optimal translation initiation(ATG) site was created based on the consensus sequence of Joshi (1987)as shown in FIG. 6a. Plants transformed with this modified Bt ICP geneshow significantly higher Bt ICP expression levels.

In accordance with this invention, all or part of modified Bt ICP geneof the invention can be stably inserted in a conventional manner intothe nuclear genome of a plant cell, and the so-transformed plant cellcan be used to produce a transgenic plant showing improved expression ofthe Bt ICP gene. In this regard, a disarmed Ti-plasmid, containing themodified Bt ICP gene, in Agrobacterium (e.g., A. tumefaciens) can beused to transform a plant cell using the procedures described, forexample, in EP 116,718 and EP 270,822, PCT publication 84/02913, EPA87400544.0 and Gould et al. (1991) (which are incorporated herein byreference). Preferred Ti-plasmid vectors contain the foreign DNAsequence between the border sequence, or at least located to the left ofthe right border sequence, of the T-DNA of the Ti-plasmid. Of course,other types of vectors can be used to transform the plant cell, usingprocedures such as direct gene transfer (as described, for example, inEP 233,247), pollen mediated transformation (as described, for example,in EP 270,356, PCT publication WO 85/01856, and U.S. Pat. No.4,684,611), plant RNA virus-mediated transformation (as described, forexample, in EP 67,553 and U.S. Pat. No. 4,407,956), liposome-mediatedtransformation (as described, for example, in U.S. Pat. No. 4,536,475)and other methods such as the recently described methods fortransforming certain lines of corn (Fromm et al., 1990; Gordon-Kamm etal., 1990).

Preferably, the modified Bt ICP gene is inserted in a plant genomedownstream of, and under the control of, a promoter which can direct theexpression of the gene in the plant cells. Preferred promoters include,but are not limited to, the strong constitutive 35S promoter (Odell etal., 1985) of cauliflower mosaic virusl; 35S promoters have beenobtained from different isolates (Hull and Howell, Virology 86, 482-493(1987)). Other preferred promoters include the TR1' promoter and theTR2' promoter (Velten et al., 1984). Alternatively, a promoter can beutilized which is not constitutive but rather is specific for one ormore tissues or organs. For example, the modified Bt ICP gene can beselectively expressed in the green tissues of a plant by placing thegene under the control of a light-inducible promoter such as thepromoter of the ribulose-1,5-phosphate-carboxylase small subunit gene asdescribed in EPA 86300291.1. Another alternative is to use a promoterwhose expression is inducible by temperature or chemical factors.

It is also preferred that the modified Bt ICP gene be inserted upstreamof suitable 3' transcription regulation signals (i.e., transcript 3' endformation and polyadenylation signals) such as the 3' untranslated endof the octopine synthase gene (Gielen et al., 1984) or T-DNA gene 7(Velten and Schell, 1985).

The resulting transformed plant of this invention shows improvedexpression of the modified Bt ICP gene and hence is characterized by theproduction of high levels of Bt ICP. Such a plant can be used in aconventional breeding scheme to produce more transformed plants with thesame improved insect-resistance characteristics or to introduce themodified Bt ICP gene into other varieties of the same or related plantspecies. Seeds, which are obtained from the transformed plants, containthe modified BtICP gene as a stable genomic insert.

Furthermore, at least two modified BtICP genes, coding for twonon-competitively binding anti-Lepidopteran or anti-Coleopteran Bt ICPS,can be cloned into a plant expression vector (EPA 89401499.2). Plantstransformed with such a vector are characterized by the simultaneousexpression of at least two modified BtICP genes. The resultingtransgenic plant is particularly useful to prevent or delay developmentof resistance to Bt ICP of insects feeding on the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Comparison of the transcription initiation frequency of RNApolymerase II complexes in nuclei of N28-220. Hybridisation efficienciesof labeled nptII mRNA and Bt ICP mRNA with their complementary DNAcounterparts present on a Southern blot were compared. DNA fragmentswere obtained from a digest of plasmid pGSH163. A schematic view of theregion is given. The lengths of the fragments blotted on Hybond-H filter(1), the homologous genes on plasmid pGSH163 (2), and the densitometricvalues (3) are as follows:

    ______________________________________    Digest:     1           2       3    ______________________________________    BamHI/HindIII                2358        neo     12386                1695        bt2      6565                 154        bt2     --                6250        vector  --    ______________________________________

FIGS. 2A-2C.--FIG. 2a--Determination of the distribution of the RNApolymerase II complexes on the Bt ICP coding sequence in nuclei ofN28-220. The hybridisation of labeled RNA prepared by nuclear run onwith DNA fragments of the Bt ICP coding sequence was quantitated. Therestriction fragments and scanning values are given in the table (FIG.2A-2) and figure (FIG. 2A-1). The scanning value is proportional to "X",the size of the DNA fragment and the # UTP per RNA fragment hybridising."X" is directly proportional to the number of RNA polymerases passingthrough the DNA fragment. "X" is proportional to the scanning valuedivided by the number of UTPS. The X values of the different restrictionfragments are shown in the figure. In this regard, conversion of thedifferent densitometric values into relative hybridisation efficienciesby normalising the values of the number of dATPs present in the DNAfragment, complementary to the hybridising RNA, generates the value "X"."X" is a relative measure of the number and the length of the extensionof the transcripts. "X" thus reflects the number of RNA polymerasestranscribing a specific DNA sequence and their elongation rate. DNAfragments present on the Southern digests of plasmid DNA of plant vectorpGSH163 each have the following lengths of fragments blotted on Hybond-Nfilter (1), homologous genes on plasmid pGSH163 (2) and densitometricvalues (3):______________________________________Digest: 1 23______________________________________BamHI/EcoRI 8877 neo 15333 726bt2(2) 2926 583 bt2(3) 635 271 bt2(1) --BamHI/EcoRV 8887 neo 15182 84bt2 2466 729 bt2 1102BamHI/HindIII 6250 -- -- 2358 neo 12386 1695 bt26565 154 -- --BamHI/SacI 8053 neo 14194 1353 bt2(1) 4572 1051 bt2(2)615XmnI 4973 neo 13219 2107 -- -- 1401 -- -- 729 bt2(3) 736 628 bt2(2)1817 305 bt2(4) -- 188 bt2(5) -- 120 bt2(1)--______________________________________

FIG. 2b--Schematic view of nine bt884 DNA fragments that were insertedinto the polylinker of M13 vectors, MP18 and MP19 (Yanisch-Perron etal., 1985). The Bt ICP coding sequence is shown from AUG to 1600nucleotides downstream. The relevant restriction sites and sizes of theDNA fragments are indicated. The nucleotide numbering is relative to theAUG. The subclones were named pJD71, pJD72, pJD73, etc. (to pJD79), asindicated. The inserts were oriented into the M13 vector such thatsingle standed M13 carried the fragments of the Bt ICP coding sequencein an antisense orientation.

FIG. 2c--Schematic representation of three nuclear run-on analyses withN28-220 nuclei as described by Cox and Goldberg (1988). Assays wereperformed for periods of 5, 10 and 30 minutes. The labeled nuclear RNAwas allowed to hybridize with 5 μg of single stranded pJD71-pJD79 andMP18 DNA, which were immobilised on nylon membranes. The membranes wereautoradiographed, and densitometric values were obtained by scanning theautoradiographs. The abscissa shows the nucleotide position relative tothe AUG of the Bt (i.e., bt2) coding sequence. The center of each of thesingle stranded Bt DNA fragments is indicated in the graph. The ordinategives the relative hybridisation signal for each fragment corrected forthe number of dATPs in the fragment and adjusted to 100% for the valueof pJD71 for each of the three incubation periods. All values arecorrected for non-specific hybridisation to single stranded MP18 DNA.The relative values are a measure for the reactivation of bt mRNAsynthesis by RNA polymerase II. The assay does not distinguish betweenthe number of mRNA extensions and the length of mRNA extensions.

FIGS. 3A-3D--Construction of deletion mutants of the bt860-neo gene tomeasure the effect on cytoplasmic Bt ICP mRNA levels. The parentalvector pVE36 is shown. (FIG. 3A)

The following deletion mutants were generated:

1. PJD50 (FIG. 3B): pJD50 was derived from pVE36 by digesting with BamHIand SphI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

2. PJD51 (FIG. 3B): pJD51 was derived from pVE36 by digesting with SpeIand SphI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

3. PJD52 (FIG. 3B): pJD52 was derived from pVE36 by digesting with EcoRVand SphI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

4. PJD53 (FIG. 3B): pJD53 was derived from pVE36 by digesting with XcaIand SphI. The 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

5. PJD54 (FIG. 3B): pJD54 was derived from pVE36 by digesting with AflIIand SphI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

6. PJD55 (FIG. 3B): pJD55 was derived from pVE36 by digesting with ClaIand SphI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

7. PJD56 (FIG. 3B): pJD56 was derived from pVE36 by digesting with XhoIand SphI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

8. PJD57 (FIG. 3B): pJD57 was derived from pVE36 by digesting with AflIIand BamHI. The 5' and 3' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

9. PJD58 (FIG. 3B): pJD58 was derived from pVE36 by digesting with XcaIand BamHI. The 5' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

10. PJD59 (FIG. 3B): pJD59 was derived from pVE36 by digesting withEcoRV and BamHI. The 5' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

11. PJD60 (FIG. 3B): pJD60 was derived from pVE36 by digesting with SpeIand BamHI. The 5' protruding ends were filled in with Klenow DNApolymerase I enzyme. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

12. PJD61 (FIG. 3C): PJD61 was derived from PJD50. PVE36 was digestedwith XbaI and filled in with Klenow polymerase I. PJD50 was linearizedwith BamHI and filled in with Klenow polymerase I. The 375 bp XbaIfragment of PVE36 was ligated in the filled in BamHI of pJD50. Theligation mixture was used to transform MC1061 cells. Transformants wereselected for amp^(r) phenotype.

13. PJD62 (FIG. 3C): PJD62 was derived from PJD50. PVE36 was digestedwith XcaI and EcoRV. PJD50 was linearized with BamHI and filled in withKlenow polymerase I. The 367 bp XcaI-EcoRV fragment of PVE36 was ligatedin the filled in BamHI of pJD50. The ligation mixture was used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

14. PJD63 (FIG. 3C): PJD63 was derived from PJD50. PVE36 was digestedwith XcaI and EcoRV. PJD50 was linearized with BamHI and filled in withKlenow polymerase I. The 474 bp XcaI-EcoRV fragment of PVE36 was ligatedin the filled in BamHI of pJD50. The ligation mixture was used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

15. PJD64 (FIG. 3C): PJD64 was derived from PJD50. PVE36 was digestedwith EcoRI and EcoRV and filled in with Klenow polymerase I. PJD50 waslinearized with BamHI and filled in with Klenow polymerase I. The 458 bpEcoRI-EcoRV fragment of PVE36 was ligated in the filled in BamHI ofpJD50. The ligation mixture was used to transform MC1061 cells.Transformants were selected for amp^(r) phenotype.

16. PJD65 (FIG. 3C): PJD65 was derived from PJD50. PVE36 was digestedwith EcoRI and XbaI and filled in with Klenow polymerase I. PJD50 waslinearized with BamHI and filled in with Klenow polymerase I. The 327 bpEcoRI-XbaI fragment of PVE36 was ligated in the filled in BamHI ofpJD50. The ligation mixture was used to transform MC1061 cells.Transformants were selected for amp^(r) phenotype.

17. PJD66 (FIG. 3C): PJD66 was derived from PJD50. PVE36 was digestedwith SpeI and XcaI and filled in with Klenow polymerase I. PJD50waslinearized with BamHI and filled in with Klenow polymerase I. The 1021bp SpeI-XcaI fragment of PVE36 was ligated in the filled in BamHI ofpJD50. The ligation mixture was used to transform MC1061 cells.Transformants were selected for amp^(r) phenotype.

18. PPS56D1 (FIG. 3D): PPS56D1 was derived from PJD56 by digesting withEcoRV. The treated DNA was ligated and then used to transform MC1061cells. Transformants were selected for amp^(r) phenotype.

19. PPS56D2 (FIG. 3D): PPS56D2 was derived from PJD56 by digesting withXcaI and AflII. The 5' protruding ends were filled in with Klenowpolymerase I. The treated DNA was ligated and then used to transformMC1061 cells. Transformants were selected for amp^(r) phenotype.

20. PPS56D3 (FIG. 3D): PPS56D3 was derived from PJD56 by digesting withSpeI and EcoRV. The 5' protruding ends were filled in with Klenowpolymerase I. The treated DNA was ligated and then used to transformMC1061 cells. Transformants were selected for amp^(r) phenotype.

21. PPS56D4 (FIG. 3D): PPS56D4 was derived from PJD56 by digesting withXcaI and partially with EcoRV. The treated DNA was ligated and then usedto transform MC1061 cells. Transformants were selected for amp^(r)phenotype.

22. PPS56D6 (FIG. 3D): PPS56D6 was derived from PJD56 by digesting withSpeI and partially with EcoRV. The 5' protruding ends were filled inwith Klenow polymerase I. The treated DNA was ligated and then used totransform MC1061 cells. Transformants were selected for amp^(r)phenotype.

23. PPS56D7 (FIG. 3D): PPS56D7 was derived from PJD56 by digesting withSpeI and XcaI. The 5' protruding ends were filled in with Klenowpolymerase I. The treated DNA was ligated and then used to transformMC1061 cells. Transformants were selected for amp^(r) phenotype.

24. PPS56D8 (FIG. 3D): PPS56D8 was derived from PPS56D2 by digestingwith SpeI and partially with EcoRV. The 5' protruding ends were filledin with Klenow polymerase I. The treated DNA was ligated and then usedto transform MC1061 cells. Transformants were selected for amp^(r)phenotype.

FIG. 4--Effect of deletions in the Bt ICP coding sequence on cytoplasmicBt ICP mRNA levels. The cytoplasmic mRNA levels specified by theinvariable cat reference gene and the different Bt ICP deletion mutantsdescribed in FIG. 3 are listed in the table. The measurements wereconverted into relative Bt ICP mRNA abundances. Bt ICP and cat mRNAquantizations were done as described by Cornelissen (1989). Total RNAwas slot blotted and hybridised with radioactively labeled RNAcomplementary to the neo and cat coding sequences. Values werequantitated with the aid of calibration curves of cold cat and Bt ICPriboprobe transcripts.

FIG. 5--Relative transcript levels produced by the deletion derivativesof pVE36.

FIGS. 6A-6C.--FIG. 6a--Schematic presentation of the synthetic DNAsequences used to introduce a N-terminal deletion and a change of thecodons 29, 30 and 31 of the bt2 coding sequence. The oligo nucleotideswere annealed according to Engler et al. (1988) and cloned into theBstXI restriction site of plasmid pVE36, yielding pPSO27. The 7360 bpfragment of pPSO27was ligated to the the 1177bp ClaI restrictionfragment of pVE36, yielding plasmid pPSO28. pPSO28 is identical to pVE36apart for the N-terminal modification.

FIG. 6b--Schematic presentation of the synthetic DNA sequences used tointroduce an internal modification into the bt2 coding sequence. Theoligonucleotides (FIG. 6B-1) were annealed and ligated (FIG. 6B-2 and6B-3) as described by Engler et al. (1988) and the resultingconcatemeric DNA fragment was cut with the restriction enzymes XbaI andEcoRI to release the modified 327 bp XbaI-EcoRI restriction fragment.This fragment was ligated into the 3530 bp EcoRI-XbaI fragment of pPSO23which is a pUC19 derivative (Yanisch-Perron et al., 1985) that carriesthe 1533 bp AflII (filled in) BamHI fragment of pVE36 in the HindIII(filled in) BamHI site of pUC19, resulting in plasmid pPSO24. PlasmidpPSO24 was linearised by digestion with restriction enzyme XbaI and the375 bp XbaI restriction fragment of pPSO23 was introduced resulting inpPSO25. The 1177 bp ClaI fragment of pPSO25 was introduced in the 7360bp ClaI restriction fragment of pPSO27 yielding pPSO29. pPSO29 isidentical to pVE36 but carries both the amino-terminal modification andthe internal modification of the Bt ICP coding sequence.

FIG. 6c--Nucleotide sequences 800 to 4000 of the plasmids pVE36 (FIG.6C-1) and pPSO29 (FIG. 6C-2). "x" refers to not known nucleotides.

FIGS. 7A-7B.--FIG. 7A--Schematic presentation of the effect of themutations on the AT content of the Bt ICP plant gene. (pVE36: FIG. 7A;pPSO29: FIG. 7B) The modified regions are indicated.

FIGS. 8A-8B--FIGS. 8A-1 and -2--Schematic presentation of the plasmidconstructions used in the transient expression assay. The relevant genesare indicated.

FIGS. 8B-1, -2 and -3--Accumulation profiles of CAT (Neumann et al.,1987) and the modified BtICP (Engvall and Pesce, 1978) in a typicaltransient expression assay.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise stated in the Examples, all procedures for making andmanipulating recombinant DNA are carried out by the standardizedprocedures described in Sambrook et al., Molecular Cloning--A laboratoryManual, Cold Spring Harbor Laboratory (1989).

EXAMPLES Example 1 Determination of the Efficiency of TranscriptionInitiation

The relative efficiency of RNA polymerase II complexes to initiatetranscription at chimaeric BtICP plant genes was studied, usingtransgenic plant N28-220 which is described by Vaeck et al. (1987) andcontains copies of the T-DNA of plasmid pGSH163 This T-DNA carries thechimaeric plant genes P_(TR2) bt8843'g7 and P_(TR1') neo3'ocs. Nuclei of25 g of induced leaves of N28-220 were prepared according to Cox andGoldberg (1988) and stored the nuclei at a temperature of -70° C. Thismethod causes the nascent precursor mRNA chains and the RNA polymeraseII complexes to halt while the complexes remain associated at the DNA. Abatch of these nuclei was assayed for the ability to incorporateradioactively labeled UTP as a measure for the transcriptional viabilityof the nuclei (Cox and Goldberg (1988). This incorporation could besuccessfully repressed by addition of α-amanitin to a finalconcentration of 2 μg/ml. This shows that the UTP incorporation was dueto transcript elongation by RNA polymerase II and that RNA synthesis onthe protein coding genes which are occupied by RNA polymerase II can bereactivated under the appropriate experimental conditions.

Batches of the nuclei of N28-220 were used to synthesize radioactivelylabeled RNA as described by Cox and Goldberg (1988). The radioactive RNAsynthesized is a direct representation of the distribution of the RNApolymerases II complexes on the DNA in the nuclei. As the DNA of N28-220carries two genes which can be assayed, namely the chimaeric neo geneand the chimaeric Bt ICP gene, it is possible to compare thedistribution of RNA polymerase II complexes on these two genes. To thisend, the radioactive RNA was extracted from the nuclei according to Coxand Goldberg (1988) and used as a probe in a conventional Southernhybridisation. The Southern blot contained DNA fragments carrying the BtICP and neo coding sequences in a molar excess relative to the neo andBt ICP RNA species present in the radioactive probe. A detaileddescription of the Southern blot is given in FIG. 1. The hybridisationexperiment resulted in hybridisation signals to both the neo and Bt ICPcoding sequences (FIG. 1). Densitometric scanning showed that theintensity of the hybridisation signal to the neo and Bt ICP codingregions was nearly identical. This result implies that the number oftranscripts initiating from the TR dual promoter is about similar inboth directions. As in plant N28-220 the cytoplasmic neo mRNA level isseveral magnitudes higher than that of Bt ICP; this shows that the BtICP coding sequence indeed negatively controls accumulation ofcytoplasmic Bt ICP mRNA, but that this phenomenon is not due to adominant negative effect on transcription initiation of the chimaeric BtICP plant gene.

Example 2 Transcription Elongation

The relative distribution of RNA polymerase II complexes on the Bt ICPplant genes present in transgenic plant N28-220 which is described byVaeck et al. (1987) was investigated. To this end, a second experimentwas carried out with batches of the nuclei of N28-220 described inExample 1.

The nuclei were incubated as described by Cox and Goldberg (1988) tosynthesize radioactively labeled RNA. The radioactive RNA was extractedas described previously to provide a probe for a Southern hybridisation.The Southern blot prepared for this experiment contained severalfragments of the Bt ICP coding sequence in molar excess relative to thecomplementary RNA present in the probe. The rationale of the experimentwas that if the RNA polymerase II complexes were equally distributedover the Bt ICP coding region, the hybridisation with the different BtICP DNA fragments present on the Southern blot would be proportional tothe size and DATP content of the different fragments. A detaileddescription of the DNA fragments present on the Southern is given inFIG. 2a. The hybridisation of the radioactive RNA extracted from thenuclei of N28-220 with the Southern revealed that the complete Bt ICPcoding sequence as present in N28-220 is transcribed by RNA polymeraseII.

Quantification of the hybridisation signals by densitometric scanning ofthe autoradiogram showed that more radioactively labeled RNA washybridising with DNA fragments representing Bt ICP sequences located 5'on the Bt ICP coding sequence than with Bt ICP sequences located 3' onthe Bt ICP coding sequence. The actual values are given in FIG. 2a. Thisin vitro experiment demonstrates that in vivo the RNA polymerases arenot evenly distributed over the Bt ICP coding sequence.

The site(s) involved in reducing the RNA polymerase II elongation werethen determined more accurately. Nine M13 derivatives were made thatcarry overlapping fragments of the Bt2 coding sequence spanning theregion from the AUG to 1584 nucleotides downstream. The inserts wereoriented into the vector such that, in single stranded M13 derivatives,the Bt sequences were complementary to the Bt transcript. A schematicview of the M13 clones is given in FIG. 2b.

A molar excess of each single stranded anti-Bt DNA was bound to nylonfilters to serve as a DNA target for hybridisation with labeled RNAprepared from nuclear run-on assays with N28-220 nuclei as described byCox and Goldberg (1988). Three nuclear run-ons that differed only intheir time period of incubation were carried out simultaneously. Theincubation time determines the length of extension of the nascent mRNAchain. Shorter incubation periods give a more accurate view of theposition of the RNA polymerase II complexes relative to the substrateDNA and their ability to elongate at the moment of the start ofincubation. Hence, the shorter the in vitro incubation period, the moreaccurate the view in predicting the in vivo situation.

The results are shown in FIG. 2c. The data for the minute incubationshow that, in vivo, at a very discrete inhibitory zone along the bt2coding sequence, one or more factors interfere with transcriptelongation and that such factor(s) remain present in such inhibitoryzone during the course of the in vitro mRNA extension reaction.Increased incubation periods show that, on a subset of DNA templates,RNA synthesis resumed downstream of such inhibitory zone in this assaywithout significantly removing the inhibition in the inhibitory zoneitself. In this regard, the data indicate that:

1. The inhibitory zone causes the RNA polymerases to pause and not toterminate.

2. This pause is only transitory for a small fraction of the Bt DNAtemplates which were used.

3. The continued RNA polymerase elongation, downstream of the inhibitoryzone, is done by a large number of polymerases on the relatively smallfraction of the Bt DNA templates.

It is believed, therefore, that low cytoplasmic Bt mRNA levels are dueat least in part to inefficient production of precursor mRNA caused byinefficient elongation of a nascent transcript and/or stalling of RNApolymerase II complexes from transcribing at an inhibitory zone.

The inhibitory zone was assayed for its ability to interact withproteins present in nuclei of tobacco protoplasts. A crude nuclearextract was prepared from tobacco SR1 leaf protoplasts according toLuthe and Quatrano (1980) and used for filter binding assay essentiallyas described by Diffley and Stillman (1986). 100 ng samples of proteinextract were mixed with different amounts of radioactively labeled 532bp XbaI-AccI bt884 DNA fragment, ranging from 0 to 1670 picomolar, in afinal volume of 0.150 ml binding buffer (10 mM Tris pH 7.5, 50 mM NaCl,1 mM DTT, 1 mM EDTA and 5% glycerol). After 45 minutes incubation atroom temperature, the samples were filtered through an alkali-washednitrocellulose membrane and washed twice with 0.150 ml of an ice-coldsolution containing 10 mM Tris pH 7.5, 50 mM NaCl and 1 mM EDTA. Theretention of DNA-protein complex was quantified by scintillationcounting and revealed that the binding had a dissociation constant inthe 100 picomolar range. The binding was not affected by preincubationof the nuclear extract with a molecular excess of a specific competitorDNA.

Example 3 Construction of Deletion Mutants

The previous two examples demonstrate that the Bt ICP coding sequence ina chimaeric plant gene negatively affects the cytoplasmic Bt ICP mRNAlevel directed by the chimaeric plant gene. It is shown that thisnegative control is not at the level of transcription initiation but atleast in part due to a reduced ability of RNA polymerase II to generateprecursor Bt ICP mRNA. A deletion analysis of the chimaeric Bt ICP plantgene was performed to identify whether impaired transcription elongationis the exclusive mechanism by which the Bt ICP sequence interferes withgene expression. The rationale of the experiment is that theintroduction of specific deletions in the Bt ICP coding region couldremove or inactivate the sequence element(s) responsible for thenegative control. As a result such mutant gene would direct an increasedlevel of cytoplasmic mRNA. This method can therefore be used to map andidentify the sequence(s) involved in the negative control.

To perform this analysis, a deletion series of the bt860-neo gene (Vaecket al., 1987) was made. FIG. 3 gives a schematic representation. Theresultant deletion derivatives do not specify a Bt ICP and therefore areassayed at RNA level only. In order to obtain accurate Bt ICP mRNAconcentration values, the deletion mutants were compared in a transientexpression system using tobacco leaf protoplasts of SR1 (Cornelissen andVandewiele, 1989). The relative mRNA abundances were calculated using acorrection factor provided by the mRNA level specified by the catreference gene present on the same plasmid as the mutant Bt ICP gene.Four hours after introduction of the genes the tobacco leaf protoplastswere harvested, and total RNA was prepared and analysed (FIG. 4).

The mutants nos. 50-60 (FIG. 3) show that progressive deletions of thecarboxy-terminal part or the amino-terminal part of the Bt ICP codingsequence result in a gradually increasing neo transcript level. As thereare not very abrupt changes in transcript levels, these results suggestthat the low transcript level produced by full length Bt ICP genes iscontrolled by a number of signals. Deletions within the Bt ICP codingsequence indeed did not localise a specific sequence element which, byitself, is responsible for the low Bt ICP mRNA level. Similarly, cloningof fragments of the Bt ICP coding sequence in pJD50 (FIG. 3) did notallow identification of such a region.

The relative transcript levels were plotted against the length of the BtICP sequence present in the different deletion derivatives. FIG. 5suggests that hybrid Bt ICP-neo transcript levels drop with increasinglength of the Bt ICP sequence. In this respect, the mutants nos. 61-66(FIG. 3) form an exception as they show in average a low transcriptlevel relative to the length of the Bt ICP sequence.

These results show that the low transcript levels of Bt ICP plant genesin tobacco are not exclusively due to an impaired elongation of thenascent transcript but that a number of signals operate to cause areduced expression capacity of the chimaeric Bt ICP gene.

Example 4

To determine whether cytoplasmic events are important in causinginefficient expression of the bt2 gene in plants, the following test wascarried out. Cytoplasmic bt2 mRNA steady state levels in transgenic leafprotoplasts of N28-220 are normally found to be below 1 transcript percell. The steady state level is determined by, and is proportional to,the number of bt2 transcripts entering per time unit the cytoplasm andthe cytoplasmic half-life of the transcript. When steady state levelsare achieved, the absolute numbers of transcripts entering and leavingthe cytoplasmic bt2 mRNA pool are equal. Therefore, the cytoplasmichalf-life and cytoplasmic steady state level of the bt2 transcript willreveal whether its cytoplasmic steady state level is due to a relativelylow import of bt2 transcript, a relatively high turnover (i.e.,conversion to a protein) rate, or a combination of both.

The cytoplasmic turnover of bt884 transcripts was determined accordingto Gallie et al. (1989). A capped and polyadenylated synthetic bt884mRNA was produced in vitro according to protocols of Promega Corporation(Madison, Wis., USA) and introduced into tobacco leaf protoplastssimultaneously with a synthetic bar (De Block et al., 1987) mRNA. Thetwo synthetic transcripts differed only in their coding sequences. Atvarious times after RNA delivery, samples were taken, and total RNA wasisolated. Northern analyses revealed that the half-lives (T 1/2) of thesynthetic bt884 and bar transcripts were about 8±3 hours and 5±2 hours,respectively. See Table 1, below. These data show that the bt884 codingsequence, more particularly the bt884 codon usage and the AU-rich motifsin the bt884 coding sequence, do not render the bt884 mRNA more unstablethan the bar mRNA which is known to accumulate in the cytoplasm to about1000 transcripts per tobacco leaf protoplast (calculated fromCornelissen, 1989). The low cytoplasmic steady state level of the bt884transcripts is, therefore, caused by a lack of import of transcriptsinto the cytoplasm. Thus, the expression defect of the bt884 gene has tobe restored by introduction of modifications in the bt884 codingsequence that improve the expression pathway in the nucleus.

Expression of the bt14, bt15 and bt18 genes in tobacco revealed thatthese genes also direct low cytoplasmic mRNA steady state levels.Therefore, a similar analysis was carried out with synthetic bt14, bt15and bt18 transcripts to identify whether the expression defect had acytoplasmic or nuclear character. Table 1, below, shows that all threetranscripts behave as stable mRNAs in the cytoplasm of tobacco leafprotoplasts. Therefore, bt14, bt15 and bt18 genes, like the bt884 gene,must be deficient in exporting high levels of bt transcript to thecytoplasm, and to improve the expression of such genes, it is necessaryto modify their coding sequences so that nuclear events, which interferewith efficient gene expression, are avoided or ameliorated.

                  TABLE 1    ______________________________________    Half-life determination of synthetic bt and bar mRNAs    in Nicotiana tabacum cv. Petite Havanna SR1 leaf    protoplasts                      T1/2               T1/2    Example 1.sup.st mRNA                      (Hours)    2.sup.nd mRNA                                         (Hours)    ______________________________________    A       bt884      8 +/- 3   bar     5 +/- 2    B       bt14       7 +/- 2   bar     6 +/- 3    C       bt15      12 +/- 5   bar     21 +/- 12    D       bt18      10 +/- 5   bar     12 +/- 5    ______________________________________

Legend

The synthetic bar transcripts had a length of 783 bases and included acap, the TMV leader (77 bases, Danthinne and Van Emmelo, 1990), the barcoding sequence (552 bases; De Block et al., 1987), a trailer of 52nucleotides consisting of the bases GAUCA CGCGA AUU and 39 bases fromthe pGEM-3Z (Promega) polylinker (KpnI (T4 DNA pol.)-HindIII (T4 DNApol.), and a poly(A) of the composition (A)₃₃ G(A)₃₂ G(A)₃₂, followed bythe nucleotides GCU.

The synthetic bt884 transcripts had a length of 2066 bases and includeda cap, the TMV leader (77 bases), the bt884 coding sequence followed bythe trailer until the Klenow treated PstI site (1843 nucleotides), thetrailer continued with AAUUC CGGGG AUCAA UU, 39 bases of the pGEM-3Zpolylinker and the (A)₃₃ G(A)₃₂ G(A)₂₁ poly(A), followed by thenucleotides CG.

The synthetic bt14 transcripts had a length of 2289 bases and included acap, the TMV leader (77 bases), the bt14 coding sequence till the Klenowtreated BclI site (2023 bases), plus 26 supplementary nucleotides CG UCGACC UGC AGC CAA GCU UGC UGA, a trailer starting with UUGAU UGACC GGAUCCGGCU CUAGA AUU, followed by 39 bases of the pGEM-3Z polylinker, and the(A)₃₃ G(A)₃₂ G(A)₂₁ poly(A), followed by the nucleotides CGGUA CCC.

The synthetic bt15 transcripts had a length of 2198 bases and included acap, the TMV leader (77 bases) the bt15 coding sequence as in pVE35 (PCTpublication WO90/15139) followed by the trailer till the Klenow treatedBamHI site (1989bases), the trailer then continued with AAUU, 39 basesof the pGEM-3Z polylinker and the (A)₃₃ G(A)₃₂ G(A)₂₁ poly(A), followedby the nucleotides CG.

The synthetic bt18 transcripts had a length of 2184 bases and included acap, the TMV leader (77 bases) the bt18 coding sequence until the Klenowtreated BcLI site (1918 bases), followed by 26 nucleotides until thetranslation stop CG UCG ACC UGC AGC CAA GCU UGC UGA, a trailer startingwith UUGAU UGACC GGAUC GAUCC GGCUC AGAUC AAUU, 39 bases of the pGEM-3Zpolylinker and the (A)₃₃ G(A)₃₂ G(A)₂₁ poly(A), followed by thenucleotides CG.

Example 5 Construction of Modified Bt ICP Genes

Examples 1-4 show that the expression in a plant of a Bt ICP gene isnegatively affected by the Bt ICP coding sequence at bothtranscriptional and post-transcriptional levels, but principally bynuclear events. These examples also show that the control of expressionis not confined to a specific DNA sequence within the Bt ICP codingsequence. Instead, the negative effect on gene expression is anintrinsic property of the Bt ICP coding sequence. On this basis, it isbelieved that, by directed change of the DNA sequence of the Bt ICPcoding region, an improvement of gene expression will occur. Theimprovement will be of a cumulative type as the negative influence ofthe Bt ICP coding region is spread over the complete coding sequence.Similarly, an improvement of gene expression will be obtained byreduction of the length of the Bt ICP coding sequence. This improvementwill have a cumulative effect if used in combination with modificationsof the Bt ICP coding region.

Therefore, two types of modifications were introduced into a Bt ICP(i.e., bt2) coding sequence which, as will be shown, indeed resulted ina significant increase in Bt ICP plant gene expression. First, the DNAsequence was modified in the central region of the toxic core fragmentof the Bt ICP as transcription elongation is impaired in this region.Secondly, the length of the Bt ICP coding sequence was reduced as thenegative influence is proportional to the length of the Bt ICP codingsequence. A detailed description of the mutations is given in FIGS. 6a,band c. As shown in FIG. 7, the modifications change the AT-content ofthe chimaeric Bt ICP gene significantly. The modifications change theprimary DNA structure of the Bt ICP coding sequence without affectingthe amino acid sequence of the encoded protein. It is evident that, ifmore DNA mutations were to be introduced into the Bt ICP codingsequence, a further improvement of gene expression would be obtained.

To determine the effect of the modifications, the expression propertiesof the modified BtICP gene and the parental bt860-neo gene were comparedin a transient expression system as described by Cornelissen andVandewiele (1989) and Denecke et al. (1989). Basically, the accumulationprofiles of the genes under study were compared by relating theirprofiles to the profile of a reference gene present in the sameexperiment. FIG. 8a shows the vectors used in the assay, and FIG. 8bshows that the accumulation of the reference CAT protein is nearlyidentical in both experiments. It is not possible to measure theaccumulation of Bt ICP encoded by the parental bt860-neo gene, but themodified Bt ICP gene clearly directs an increased synthesis of Bt ICP.

These results demonstrate that mutation of the Bt ICP coding sequencerelieves the negative influence of the Bt ICP coding sequence on theexpression of a Bt ICP plant gene.

Example 6 Cloning and Expression of Modified BT ICP Genes in Tobacco andPotato Plants

Using the procedures described in U.S. patent application Ser. No.821,582, filed Jan. 22, 1986, and EPA 86300291.1, EPA 88402115.5 and EPA89400428.2, the modified Bt ICP (i.e., bt2) genes of FIGS. 6 and 7 areinserted into the intermediate T-DNA vector, pGSH1160 (Deblaere et al.,1988) between the vector's T-DNA terminal border repeat sequences.

To obtain significant expression in plants, the modified Bt ICP genesare placed under the control of the strong TR2' promoter (Velten et al.,1984) and are fused to the transcript 3' end formation andpolyadenylation signals of the T-DNA gene 7 (Velten and Schell, 1985).

In addition, the translation initiation context or site are changed inaccordance with the Joshi consensus sequence (Joshi, 1987) in order tooptimize the translation initiation in plant cells. To this end, anoligo duplex (FIGS. 6a and 6b) is introduced to create the followingsequence at translation initiation site: AAAACCATGGCT. In this way, anadditional codon (i.e., GCT) coding for alanine is introduced.Additionally, KpnI and BstXI sites are created upstream of the ATGtranslation initiation codon.

Using standard procedures (Deblaere et al., 1985), the intermediateplant expression vectors, containing the modified BtICP gene, aretransferred into the Agrobacterium strain C58C1 Rif^(R) (U.S. patentapplication Ser. No. 821,582; EPA 86300291.1) carrying the disarmedTi-plasmid pGV2260 (Vaeck et al., 1987). Selection for spectinomycinresistance yields cointegrated plasmids, consisting of pGV2260 and therespective intermediate plant expression vectors. Each of theserecombinant Agrobacterium strains is then used to transform differenttobacco plant cells (Nicotiania tabacum) and potato plant cells (Solanumtuberosum) so that the modified Bt ICP genes are contained in, andexpressed by, different tobacco and potato plant cells.

The transgenic tobacco plants containing the modified Bt ICP genes areanalyzed with an ELISA assay. These plants are characterized by asignificant increase in levels of Bt (Bt2) proteins, compared to atransgenic tobacco plant containing a non-modified Bt ICP (bt2) gene.

The insecticidal activity of the expression products of the modified BtICP (bt2) genes in leaves of transformed tobacco and potato plants isevaluated by recording the growth rate and mortality of larvae ofTobacco hornworm (Manduca sexta), Tobacco budworm (Heliotis virescens)and potato tubermoth (Phthorimaea operculella) fed on leaves of thesetwo types of plants. These results are compared with the growth rate oflarvae fed leaves from tobacco and potato plants transformed with theunmodified or parental Bt ICP (bt2) gene and from untransformed potatoand tobacco plants. Toxicity assays are performed as described in EPA88402115.5 and EPA 86300291.1.

A significantly higher mortality rate is obtained among larvae fed onleaves of transformed plants containing and expressing the modified BtICP genes. Tobacco and potato plants containing the modified Bt ICPgenes show considerably higher expression levels of Bt ICPs compared totobacco and potato plants containing the unmodified Bt ICP gene.

The insecticidal activity of three transgenic tobacco plants containingthe modified Bt ICP genes is determined against second and third instarlarvae of Heliothis virescens. The control plant was not transformed.The results are summarized in Table 2, below.

                  TABLE 2    ______________________________________              % mortality of insects (recorded after    Plant     5 days)    ______________________________________    Control   11    No. 1     100    No. 2     88.5    No. 3     100    ______________________________________

Needless to say, this invention is not limited to tobacco and potatoplants transformed with the modified Bt ICP gene. It includes any plant,such as tomato, alfalfa, sunflowers, corn, cotton, soybean, sugar beets,rapeseed, brassicas and other vegetables, transformed with the modifiedBt ICP gene.

Nor is the invention limited to the use of Aarobacterium tumefaciensTi-plasmids for transforming plant cells with a modified Bt ICP gene.Other known techniques for plant transformation, such as by means ofliposomes, by electroporation or by vector systems based on plantviruses or pollen, can be used for transforming monocotyledonons anddicotyledons with such a modified Bt ICP gene.

Nor is the invention limited to the bt2 gene, but rather encompasses allCry I, Cry II, CryIII and Cry IV Bt ICP genes.

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    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 23    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GAUCACGCGAAUU13    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AAUUCCGGGGAUCAAUU17    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CGUCGACCUGCAGCCAAGCUUGCUGA26    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    UUGAUUGACCGGAUCCGGCUCUAGAAUU28    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CGGUACCC8    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    AAAACCATGGCT12    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GTACCAAAACCATGGCTATCGAGACCGGTTACACCCCAATCGATATCG48    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    ATCGATTGGGGTGTAACCGGTCTCGATAGCCATGGTTTTGGTACCGAT48    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 48 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    ATCGGTACCAAAACCATGGCTATCGAGACCGGTTACACCCCAATCGAT48    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 52 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 16..51    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    ATCGGTACCAAAACCATGGCTATCGAGACCGGTTACACCCCAATCGATATC51    MetAlaIleGluThrGlyTyrThrProIleAspIle    1510    G52    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 65 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GATCCTCTAGAGACTGGATCAGGTACAACCAGTTCAGGAGGGAGTTAACCCTAACCGTGT60    TAGAC65    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 71 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    ATCGTGTCCCTATTCCCGAACTACGACAGCAGGACGTACCCAATCCGAACCGTGTCCCAG60    TTAACCAGGGA71    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 65 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GATCTACACCAACCCAGTGTTAGAGAACTTCGACGGTAGCTTCCGAGGCTCGGCTCAGGG60    CATCG65    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 65 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    AGGGAAGCATCAGGAGCCCACACTTGATGGACATCCTTAACAGCATCACCATCTACACGG60    ACGCT65    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 73 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    CACAGGGGAGAGTACTACTGGTCCGGGCACCAGATCATGGCTTCCCCTGTGGGGTTCTCG60    GGGCCAGAATTCG73    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 66 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GATCCGAATTCTGGCCCCGAGAACCCCACAGGGGAAGCCATGATCTGGTGCCCGGACCAG60    TAGTAC66    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 65 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    TCTCCCCTGTGAGCGTCCGTGTAGATGGTGATGCTGTTAAGGATGTCCATCAAGTGTGGG60    CTCCT65    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 64 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    GATGCTTCCCTCGATGCCCTGAGCCGAGCCTCGGAAGCTACCGTCGAAGTTCTCTAACAC60    TGGG64    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 71 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    TTGGTGTAGATCTCCCTGGTTAACTGGGACACGGTTCGGATTGGGTACGTCCTGCTGTCG60    TAGTTCGGGAA71    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 73 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    TAGGGACACGATGTCTAACACGGTTAGGGTTAACTCCCTCCTGAACTGGTTGTACCTGAT60    CCAGTCTCTAGAG73    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 343 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..341    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    GATCCTCTAGAGACTGGATCAGGTACAACCAGTTCAGGAGGGAGTTA47    SerSerArgAspTrpIleArgTyrAsnGlnPheArgArgGluLeu    151015    ACCCTAACCGTGTTAGACATCGTGTCCCTATTCCCGAACTACGACAGC95    ThrLeuThrValLeuAspIleValSerLeuPheProAsnTyrAspSer    202530    AGGACGTACCCAATCCGAACCGTGTCCCAGTTAACCAGGGAGATCTAC143    ArgThrTyrProIleArgThrValSerGlnLeuThrArgGluIleTyr    354045    ACCAACCCAGTGTTAGAGAACTTCGACGGTAGCTTCCGAGGCTCGGCT191    ThrAsnProValLeuGluAsnPheAspGlySerPheArgGlySerAla    505560    CAGGGCATCGAGGGAAGCATCAGGAGCCCACACTTGATGGACATCCTT239    GlnGlyIleGluGlySerIleArgSerProHisLeuMetAspIleLeu    657075    AACAGCATCACCATCTACACGGACGCTCACAGGGGAGAGTACTACTGG287    AsnSerIleThrIleTyrThrAspAlaHisArgGlyGluTyrTyrTrp    80859095    TCCGGGCACCAGATCATGGCTTCCCCTGTGGGGTTCTCGGGGCCAGAA335    SerGlyHisGlnIleMetAlaSerProValGlyPheSerGlyProGlu    100105110    TTCGGATC343    PheGly    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3201 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 2151..2155    (D) OTHER INFORMATION: /note= "Nucleotides 2151-2155    wherein N is not known."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    AAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCAAATTACCAATACATTACACTA60    GCATCTGAATTTCATAACCAATCTCGATACACCAAATCGATGGATCCCGATAACAATCCG120    AACATCAATGAATGCATTCCTTATAATTGTTTAAGTAACCCTGAAGTAGAAGTATTAGGT180    GGAGAAAGAATAGAAACTGGTTACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTT240    CTTTTGAGTGAATTTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATATGG300    GGAATTTTTGGTCCCTCTCAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAAC360    CAAAGAATAGAAGAATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAAT420    CTTTATCAAATTTACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCA480    TTAAGAGAAGAGATGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATT540    CCTCTTTTTGCAGTTCAAAATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTGCA600    AATTTACATTTATCAGTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGAT660    GCCGCGACTATCAATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGAT720    CATGCTGTACGCTGGTACAATACGGGATTAGAGCGTGTATGGGGACCGGATTCTAGAGAT780    TGGATAAGATATAATCAATTTAGAAGAGAATTAACACTAACTGTATTAGATATCGTTTCT840    CTATTTCCGAACTATGATAGTAGAACGTATCCAATTCGAACAGTTTCCCAATTAACAAGA900    GAAATTTATACAAACCCAGTATTAGAAAATTTTGATGGTAGTTTTCGAGGCTCGGCTCAG960    GGCATAGAAGGAAGTATTAGGAGTCCACATTTGATGGATATACTTAACAGTATAACCATC1020    TATACGGATGCTCATAGAGGAGAATATTATTGGTCAGGGCATCAAATAATGGCTTCTCCT1080    GTAGGGTTTTCGGGGCCAGAATTCACTTTTCCGCTATATGGAACTATGGGAAATGCAGCT1140    CCACAACAACGTATTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTATCGTCCACT1200    TTATATAGAAGACCTTTTAATATAGGGATAAATAATCAACAACTATCTGTTCTTGACGGG1260    ACAGAATTTGCTTATGGAACCTCCTCAAATTTGCCATCCGCTGTATACAGAAAAAGCGGA1320    ACGGTAGATTCGCTGGATGAAATACCGCCACAGAATAACAACGTGCCACCTAGGCAAGGA1380    TTTAGTCATCGATTAAGCCATGTTTCAATGTTTCGTTCAGGCTTTAGTAATAGTAGTGTA1440    AGTATAATAAGAGCTCCTATGTTCTCTTGGATACATCGTAGTGCTGAATTTAATAATATA1500    ATTCCTTCATCACAAATTACACAAATACCTTTAACAAAATCTACTAATCTTGGCTCTGGA1560    ACTTCTGTCGTTAAAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCT1620    GGCCAGATTTCAACCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTA1680    AGAATTCGCTACGCTTCTACCACAAATTTACAATTCCATACATCAATTGACGGAAGACCT1740    ATTAATCAGGGGAATTTTTCAGCAACTATGAGTAGTGGGAGTAATTTACAGTCCGGAAGC1800    TTTAGGACTGTAGGTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACG1860    TTAAGTGCTCATGTCTTCAATTCAGGCAATGAAGTTTATATAGATCGAATTGAATTTGTT1920    CCGGCAGAAGTAACCTTTGAGGCAGAATATGATTTAGAAAGAGCACAAAAGGCGGTGAAT1980    GAGCTGTTTACTTCTTCCAATCAAATCGGGTTAAAAACAGATGTGACGGATTATCATATT2040    GATCAAGTATCCAATTTAGTTGAGTGTTTATCTGATGAATTTTGTCTGGATGAAAAAAAA2100    GAATTGTCCGAGAAAGTCAAACATGCGAAGCGACTTAGTGATGAGCGGAANNNNNCCTCG2160    AGCTTGGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTAT2220    GACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAG2280    GGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGAC2340    GAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGAC2400    GTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTC2460    CTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGG2520    CTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAG2580    CGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCAT2640    CAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAG2700    GATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGC2760    TTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCG2820    TTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTG2880    CTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAG2940    TTCTTCTGACAGATCCCCCGATGAGCTAAGCTAGCTATATCATCAATTTATGTATTACAC3000    ATAATATCGCACTCAGTCTTTCATCTACGGCAATGTACCAGCTGATATAATCAGTTATTG3060    AAATATTTCTGAATTTAAACTTGCATCAATAAATTTATGTTTTTGCTTGGACTATAATAC3120    CTGACTTGTTATTTTATCAATAAATATTTAAACTATATTTCTTTCAAGATGGGAATTAAC3180    ATCTACAAATTGCCTTTTCTT3201    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3200 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 2078..2082    (D) OTHER INFORMATION: /note= "Nucleotides 2078-2082    wherein N is not known."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    AAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCAAATTACCAATACATTACACTA60    GCATCTGAATTTCATAACCAATCTCGATACACCAAATCGGTACCAAAACCATGGCTATCG120    AGACCGGTTACACCCCAATCGATATTTCCTTGTCGCTAACGCAATTTCTTTTGAGTGAAT180    TTGTTCCCGGTGCTGGATTTGTGTTAGGACTAGTTGATATAATATGGGGAATTTTTGGTC240    CCTCTCAATGGGACGCATTTCTTGTACAAATTGAACAGTTAATTAACCAAAGAATAGAAG300    AATTCGCTAGGAACCAAGCCATTTCTAGATTAGAAGGACTAAGCAATCTTTATCAAATTT360    ACGCAGAATCTTTTAGAGAGTGGGAAGCAGATCCTACTAATCCAGCATTAAGAGAAGAGA420    TGCGTATTCAATTCAATGACATGAACAGTGCCCTTACAACCGCTATTCCTCTTTTTGCAG480    TTCAAAATTATCAAGTTCCTCTTTTATCAGTATATGTTCAAGCTGCAAATTTACATTTAT540    CAGTTTTGAGAGATGTTTCAGTGTTTGGACAAAGGTGGGGATTTGATGCCGCGACTATCA600    ATAGTCGTTATAATGATTTAACTAGGCTTATTGGCAACTATACAGATCATGCTGTACGCT660    GGTACAATACGGGATTAGAGCGTGTATGGGGACCGGATTCTAGAGACTGGATCAGGTACA720    ACCAGTTCAGGAGGGAGTTAACCCTAACCGTGTTAGACATCGTGTCCCTATTCCCGAACT780    ACGACAGCAGGACGTACCCAATCCGAACCGTGTCCCAGTTAACCAGGGAGATCTACACCA840    ACCCAGTGTTAGAGAACTTCGACGGTAGCTTCCGAGGCTCGGCTCAGGGCATCGAGGGAA900    GCATCAGGAGCCCACACTTGATGGACATCCTTAACAGCATCACCATCTACACGGACGCTC960    ACAGGGGAGAGTACTACTGGTCCGGGCACCAGATCATGGCTTCCCCTGTGGGGTTCTCGG1020    GGCCAGAATTCACTTTTCCGCTATATGGAACTATGGGAAATGCAGCTCCACAACAACGTA1080    TTGTTGCTCAACTAGGTCAGGGCGTGTATAGAACATTATCGTCCACTTTATATAGAAGAC1140    CTTTTAATATAGGGATAAATAATCAACAACTATCTGTTCTTGACGGGACAGAATTTGCTT1200    ATGGAACCTCCTCAAATTTGCCATCCGCTGTATACAGAAAAAGCGGAACGGTAGATTCGC1260    TGGATGAAATACCGCCACAGAATAACAACGTGCCACCTAGGCAAGGATTTAGTCATCGAT1320    TAAGCCATGTTTCAATGTTTCGTTCAGGCTTTAGTAATAGTAGTGTAAGTATAATAAGAG1380    CTCCTATGTTCTCTTGGATACATCGTAGTGCTGAATTTAATAATATAATTCCTTCATCAC1440    AAATTACACAAATACCTTTAACAAAATCTACTAATCTTGGCTCTGGAACTTCTGTCGTTA1500    AAGGACCAGGATTTACAGGAGGAGATATTCTTCGAAGAACTTCACCTGGCCAGATTTCAA1560    CCTTAAGAGTAAATATTACTGCACCATTATCACAAAGATATCGGGTAAGAATTCGCTACG1620    CTTCTACCACAAATTTACAATTCCATACATCAATTGACGGAAGACCTATTAATCAGGGGA1680    ATTTTTCAGCAACTATGAGTAGTGGGAGTAATTTACAGTCCGGAAGCTTTAGGACTGTAG1740    GTTTTACTACTCCGTTTAACTTTTCAAATGGATCAAGTGTATTTACGTTAAGTGCTCATG1800    TCTTCAATTCAGGCAATGAAGTTTATATAGATCGAATTGAATTTGTTCCGGCAGAAGTAA1860    CCTTTGAGGCAGAATATGATTTAGAAAGAGCACAAAAGGCGGTGAATGAGCTGTTTACTT1920    CTTCCAATCAAATCGGGTTAAAAACAGATGTGACGGATTATCATATTGATCAAGTATCCA1980    ATTTAGTTGAGTGTTTATCTGATGAATTTTGTCTGGATGAAAAAAAAGAATTGTCCGAGA2040    AAGTCAAACATGCGAAGCGACTTAGTGATGAGCGGAANNNNNCCTCGAGCTTGGATGGAT2100    TGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAAC2160    AGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTC2220    TTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGC2280    TATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAG2340    CGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACC2400    TTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTG2460    ATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTC2520    GGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGC2580    CAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGA2640    CCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCA2700    TCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTG2760    ATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCG2820    CCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGACAGA2880    TCCCCCGATGAGCTAAGCTAGCTATATCATCAATTTATGTATTACACATAATATCGCACT2940    CAGTCTTTCATCTACGGCAATGTACCAGCTGATATAATCAGTTATTGAAATATTTCTGAA3000    TTTAAACTTGCATCAATAAATTTATGTTTTTGCTTGGACTATAATACCTGACTTGTTATT3060    TTATCAATAAATATTTAAACTATATTTCTTTCAAGATGGGAATTAACATCTACAAATTGC3120    CTTTTCTTATCGACCATGTACGGGTACCGAGCTCGAATTCCACGCAGCAGGTCTCATCAA3180    GACGATCTACCCGAGTAACA3200    __________________________________________________________________________

We claim:
 1. A process for modifying a Bt ICP gene for expression inplant cells comprising:1) identifying the first region of about 300 bpdownstream from the translation initiation site of the coding sequenceof said Bt ICP gene having a transcriptional activity which is less than25% of the transcriptional activity of a region of similar length at thetranslation initiation site of said gene, and which specifically bindsto nuclear proteins isolated from said plant cells in vitro; and 2)modifying about 3 to about 63 codons in said region by changing A or Tnucleotides to G or C nucleotides without affecting the encoded aminoacid sequence, and wherein transcription of said Bt ICP gene isincreased in said plant cells.
 2. A modified Bt ICP gene for expressionin plant cells obtained by the process of claim 1.